Disruption of vitamin A metabolism by dioxin

Vitamin A (retinoids) is a nutrient that plays a central role in development and remains essential for cell growth and differentiation throughout life. Dioxins are polychlorinated organic pollutants known to negatively affect the storage and body clearance of retinoids. This work aimed at studying mechanisms and consequences of dioxin-induced retinoid disruption in vivo. As model compound we used 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and as model species adult male Sprague-Dawley rats and young and adult male and female C57Bl/6, 129/SV, and mixed C57Bl/6-129/SV mice. From the rat studies we were able to conclude that the mechanism behind inhibition of hepatic retinoid storage and induction of renal retinoid levels previously observed, is a tissue-specific transcriptional effect of TCDD on an enzyme essential for the conversion of retinol to retinyl esters for storage, lecithin:retinol acyltransferase (LRAT). This specific effect may be the only one responsible for the loss of hepatic vitamin A, as TCDD did not affect any of the known retinyl ester hydrolases thought to be responsible for the mobilization of vitamin A from storage, or the binding protein critically involved in retinyl ester formation, CRBP I. The active form of vitamin A, the nuclear receptor ligand retinoic acid, was significantly increased in liver, kidney, and serum after TCDD exposure. This was unexpected, as symptoms of dioxin exposure has previously been compared with that in vitamin A deficiency and we found no indications or symptoms of vitamin A excess in exposed animals. This is however in consistency with earlier data describing an inhibiting effect of TCDD on retinoic acid signaling. The quick and sensitive response of retinoic acid and its metabolites to TCDD suggests a direct effect of TCDD-induced enzymes on retinoic acid metabolism. From time-course and dose-effect relationships, a role of cytochrome P4501A in the all-trans retinoic acid increase was suggested. By using young and adult transgenic mice deficient in various combinations of retinoid-related genes we then investigated the possible involvement of single retinoid receptors (RARs, RXRs) and retinol- and retinoic acid-binding proteins (CRBP I, CRABP I, CRABP II) in normal retinoid metabolism as well as dioxin toxicity. Transgenic mice lacking CRBP I displayed particularly low levels of total hepatic vitamin A, retinol, and all-trans-retinoic acid, despite adequate dietary supply of vitamin A and normal body weight. Higher levels of 9-cis-4-oxo-13,14-dihydro-retinoic acid in liver were also found in mice lacking intracellular retinoid-binding proteins. We concluded that CRBP I is required for a normal retinoic acid homeostasis. Although speculated in the literature, this role of CRBP I has never been demonstrated before. CRBP I -/- mice bones had lower bone density (assessed by perpheral quantitative computed tomography) and decreased bone strength (assessed by three-point-bending test or axial loading test). However, the bone phenotype was not similar to vitamin A-deficient bone; PQCT measurements of cortical (hard) and trabecular (spongy) bone tissue in the long bones (tibia, femur, and femoral neck), as well as bone biomechanical analyses, showed that adult CRBP I -/- mice have a bone status that shows more similarities to a bone exposed to excess vitamin A, i.e. shorter, thinner, and weaker bones with lower mineral density. Thus, regarding the bone, our data suggested that the CRBP I -/- mouse has not encountered vitamin A deficiency during the life stages essential for the structure of the bone as adult. When exposed to TCDD, CRBP I -/- mice responded in a similar way as has been previously observed when these mice are fed a vitamin A-deficient diet, i.e. a total depletion of retinoid stores, hepatic all-trans-retinoic acid levels were unchanged by TCDD of all mutant and wild-type mice, whereas 9-cis-4-oxo-13,14-dihydroxy-retinoic acid levels dropped significantly. The unaffected retinoic acid levels were in contrast to the rat experiments, whereas the effect on liver 9-cis-4-oxo-13,14-dihydro-retinoic acid showed a similar pattern to rats. The effects of TCDD on bone dimensions, composition, and strength were highly dependent on whether CRBP I had been knocked out or not. A rescue effect of a disrupted CRBP I gene on TCDD-exposed bone was observed, leading one to speculate that TCDD normalized the vitamin A excess bone phenotype in the mutant. TCDD exposure decreased serum levels of the osteoclast activity biomarker Trap5b in CRBP I -/- mice, consistent with the hypothesis that dioxin induces an onset of vitamin A deficiency, which subsequently inhibits osteoclast activity and bone resorption. Furthermore, TCDD decreased serum vitamin D levels in mice, a previously unknown effect of TCDD which seemed unrelated to the effects on vitamin A. Finally, studies in the mouse suggested that the retinoid system interacts with TCDD-induced CYP1A activity, an effect previously shown only in vitro. In summary, the effects of TCDD on retinoid storage include a tissue-specific alteration in LRAT expression and activity. TCDD had a tissue- and species-dependent effect on the active retinoid metabolite all-trans-retinoic acid. The new retinoic acid metabolite, which was discovered in liver tissue at levels higher than all-trans retinoic acid, was a very sensitive indicator of TCDD exposure. CRBP I was found to play a significant role in retinoic acid homeostasis, either directly by affecting the oxidative pathway from retinol, or via its role in esterification of incoming dietary vitamin A. We observed that the bone is a target for the endocrine-disruptive actions of TCDD. The TCDD-induced effects on mouse bone growth, strength, and composition were found consistent with recent studies in the rat and did not primarily implicate altered bone resorption. Comparisons of the CRBP I -/- bone phenotype with studies of the effects of vitamin A deficiency and excess on the bone showed that both control and dioxin-disrupted bone health are negatively influenced by an altered retinoid metabolism. It is possible that adult bone health may be compromised by an excess vitamin A status during early life